Growth and differentiation of the pulmonary epithelium are essential determinants of lung function. These same processes, either as a response to lung injury, neoplastic transformation, or intrinsic abnormalities of growth and differentiation, are important factors in the pathogenesis of human lung disease. Additionally, an understanding of the biology of growth and differentiation of the pulmonary epithelium is essential for realizing the full potential of gene therapy in the lung. The long term objective of these studies is to define the molecular and cellular mechanisms regulating growth and differentiation in the proximal pulmonary epithelium. A study of the mechanisms of growth and differentiation in the developing lung will lead to important insights into the role these processes play in lung disease. The specific aims in this proposal are intended to examine the lineage relationships of cells in the developing airway epithelium and the molecular mechanisms regulating differentiation within this epithelium. These studies will utilize unique transgenic markers of primitive proximal and distal pulmonary epithelium to examine the allocation of cells to different lineages during early lung development. The role of the pulmonary mesenchyme in regulating these early developmental decisions will be examined in explanted fetal lungs by disruption of normal mesenchymal epithelial interactions. Additional insights into the lineage relationships of cells in the developing pulmonary epithelium and the role of Clara cells as progenitor cells will be gained by toxigenic ablation of cells expressing a Clara cell secretory protein-HSV thymidine kinase transgene during development. Finally, potential mediators of differentiation in the proximal pulmonary epithelium will be identified and characterized by the technique of subtractive hybridization. Fluorescence activated cell sorting will be used to obtain populations of primitive proximal and primitive distal pulmonary epithelial cells expressing a beta-galactosidase transgene. Following the construction of proximal and distal epithelial cDNA libraries, subtractive hybridization will be performed to isolate and characterize unique clones from the primitive proximal pulmonary epithelium.